The current invention relates to a global class of optical sensors that use evanescent fields to excite fluorescence from species bound to a waveguide fiber surface during assay performed in the gas phase, liquid solution, or solid environment.
The evanescent field associated with an electromagnetic field propagating in an optical waveguide typically penetrates from a few nanometers to several hundred nanometers into the medium surrounding the optical waveguide. This evanescent field can excite fluorescent materials, such as fluorophores, which will fluoresce when they are bound by molecules on or very close to the optical waveguide surface. This fluorescence can lead to an efficient and selective immunoassay or hybridization assay. For an archetypal immunoassay sensor, biological recognition (binding) of an antigen by antibodies attached to the waveguide surface with concomitant displacement of fluorescent-labeled antigen is measured as a change in fluorescence. An oligonucleotide (either RNA or DNA), to which is attached a suitable fluorophore, is recognized by a complementary oligonucleotide (either RNA or DNA).
For a hybridization assay sensor, biological recognition (binding) of an oligonucleotide is measured as a change in fluorescence. A suitable fluorophore is attached to the oligonucleotide by a complementary oligonucleotide (either RNA or DNA) that in turn is attached to the waveguide surface, with a concomitant enhancement of fluorescence.
The use of optical fibers in certain geometrical configurations for immunoassay sensors is also known. One such use of optical fibers is as waveguides that capture and conduct fluorescent radiation emitted by molecules near the optical fiber surface. However, waveguide-binding sensors of this sort that are known for use in assays of aqueous fluids have demonstrated inadequate sensitivity. Specifically, poor sensor performance is attributed at least in part to the small size of the sample being analyzed, which is typically a few monolayers deep, and to the small active surface area of the optical waveguide. These factors limit the number of fluorophores that may be excited. More serious sensor performance degradation is attributable to the effects of a weak evanescent wave that fails to excite enough fluorophores to produce detectable levels of fluorescence. In addition, the past geometries used provide inadequate coupling of the fluorescence into the waveguide for subsequent detection.
Increasing the strength of the evanescent wave penetrating into a fluid sample to be assayed increases the amount of fluorescence, thereby increasing sensor sensitivity. Each mode (low and high order) propagating in the fiber has a portion of its power in the evanescent wave. Higher order modes have a larger percentage of their power in the evanescent wave and thus make a larger contribution to power in the evanescent wave. However, these higher order modes are weakly guided and lossy and can easily leak at a discontinuity or a bending point along the waveguide. In addition, the light distribution among the many modes of a multimode fiber is a very sensitive function of the specific optical arrangement of the fiber and effects on it produced by its environment. Even small bending or other mechanical effects on the fiber change the modal intensity distribution. These effects make an evanescent field sensor based on multimode fibers noisy and less sensitive.
The use of some types of tapered optical fiber to increase the sensitivity of fiber-optic assay systems is known. For example, it is known to use optical fibers as sensors in conjunction with assays.
Evanescent sensors using acid-etched multimode fiber probes are known. Higher-order modes in these fibers have the advantage of a large number of reflections per unit length and, thus, a long interaction length with the external medium and weaker evanescent field interaction to minimize photobleaching in situations where this might be a problem. However, the use of multimode fibers has a number of disadvantages. In a multimode sensor arrangement, the lower-order modes are confined to the central core region, limiting their interaction with the surroundings. This means that the total fraction of power that interacts with the external medium depends on the distribution of modal excitations and the fraction of each mode outside the core. Also, sensors made of multimode fibers are not easily compatible with many in-line fiber components, such as fiber directional couplers, isolators, and fiber Bragg grating filters.
Tapered multimode fibers may be produced in a known way using concentrated hydrofluoric acid (HF) solution. The method involves a multi-step procedure and requires a high-precision, computer-controlled stage to lower the fiber into the acid bath over a number of time intervals. This is a time-consuming and potentially non-reproducible procedure. In the procedure, the plastic cladding of the fiber is mechanically removed. This introduces a step discontinuity between the cladded and uncladded section of the fiber. Consequently, a combination tapering procedure is required to minimize V-number mismatch and prevent excess loss of returned fluorescence radiation through the tapered fiber. Such a procedure is potentially dangerous because of the use of hazardous chemicals such as concentrated HF acid. The tapering operation must be performed under a fume hood in a clean-room environment by well trained technicians.
Use of a single-mode fiber for sensing can avoid negative aspects of multimode fibers while having advantages such as light launched into the fiber exciting only the fundamental mode, which interacts with the surrounding area in the tapering region.
Optical fibers can also be tapered by heating and drawing, whereby both the core and cladding outer diameters are decreased and ultimately merge. One such tapered optical fiber is used in microscopy applications. The tapered fiber tip is short, has an extremely small diameter (a few tens of nanometers), and has a metal coating applied over the end of the tip.
When an optical fiber is tapered by heating and drawing, both the core diameter and the cladding outer diameter are decreased. The fractional change in the core diameter is approximately equal to the fractional change in the cladding outer diameter. In other words, the cross section of the fiber changes in scale only. Significantly, the angle at which the core is tapered is substantially smaller than the taper angle xcex2, which is defined as the angle at which the drawn fiber is tapered. In one known example, the tangent of the core is only 3/125, or 2.4%, times the tangent of the taper angle xcex2. For this reason, even for relatively large values of xcex2 such as 30xc2x0 or greater, the core will have an adiabatic taper, as discussed below. When a fiber is drawn to a diameter comparable to, or smaller than, a wavelength, the boundary between core and cladding glass disappears and the structure becomes one of a glass core/fluid cladding.
In an untapered fiber, the electric component of the dielectric mode is largely confined to the core and falls to a very small amplitude, typically less than 10xe2x88x9210 times the peak amplitude, near the cladding outer surface. That is not necessarily the case in a tapered fiber. As a guided light wave propagates into the taper region, it encounters a progressively narrowing core. Eventually, the core becomes too small to substantially confine the guided mode. Instead, the light is guided by the interface between the cladding and the surrounding material, which may be air or a liquid. The core will generally be tapered at a small enough angle for the guide change to be adiabatic. By the term xe2x80x9cadiabaticxe2x80x9d it is meant that substantially all of the energy of the initial fundamental guided mode remains concentrated in a single mode, and is not coupled into other modes, particularly radiation modes, which lead to a loss of energy from the waveguide.
One potential disadvantage of fluorosensors is their use of fluorophores whose excitation and emission wavelengths are close to each other. It is very important to filter out the excitation light at the receiving port. Commonly, one or more low-pass filters (LPF) are placed in the return path to block the stray excitation light. However, commercially available dielectric LPF""s do not have a sharp enough lower wavelength spectral characteristic and therefore cannot block the stray signal aft efficiently. Furthermore, previously known bulk optics arrangement using an off-axis perforated parabolic reflector are very costly and require crucial optical alignment. Photobleaching of the fluorophore when exposed to continuous laser irradiation in many sensors remains a serious problem and severely restricts fluorosensor sensitivity. In addition, in sensors using bulk optics, the large size, number of optic components that must be carefully aligned, and overall bulk of current fluorosensors makes them unsuited for general and widespread use.
An object of the invention is to provide a detector that overcomes the above-noted deficiencies.
Another object of the invention is to provide a detector that has a variety of applications in the fields of biology, biochemistry, chemistry, pharmacology and in many clinical applications.
To these and other objects, the present invention is directed to a probe for detecting a chemical, the probe comprising: an optical fiber having a tapered portion in which a diameter of the optical fiber is reduced from a larger diameter to a smaller diameter; and a coating disposed on a surface of the tapered portion of the optical fiber, the coating having a property of binding with the chemical when brought in contact therewith. The tapered fiber is held in a mechanical holder that is specially treated to prevent its reacting with any of the material that the treated fiber is designed to detect. The holder can also be placed in or made part of a flow cell with separate ports for entry and exit of fluid. Detection by the fiber can take place as fluid streams over the sensor.
The present invention is further directed to a method of making a probe for detecting a chemical, the method comprising: (a) forming a tapered portion in an optical fiber so that a diameter of the optical fiber is reduced in the tapered portion from a larger diameter to a smaller diameter; and (b) applying a coating on a surface of the tapered portion of the optical fiber, the coating having a property of binding with the chemical when brought in contact therewith.
The detector according to the present invention uses a tapered fiber to excite fluorescence from surface bound fluorophores and to couple the fluorescence back into the fiber. The fluorescence arises from species binding on the fiber surface resulting from the interaction (chemical, biochemical, bioaffinity, or immunogenic-type) of biomolecules (ligands) with their respective binding partners. The terms xe2x80x9cligandxe2x80x9d and xe2x80x9cbinding partnerxe2x80x9d for the ligand are used to represent the two components in specific bioaffinity binding pairs, all of which are capable of recognizing and binding with the other partner in a biomolecular recognition pair. Examples of such binding pairs include: antigen-antibody, substrate-enzyme, effector-enzyme, inhibitor-enzyme, complementary nucleic acid strands (RNA or DNA), binding protein-vitamin, binding protein-nucleic acid, reactive dye-protein, reactive dye-nucleic acid, receptor-agonist or antagonist, and others.
Each of the binding partners is firmly attached to the tapered fiber surface and within the evanescent field. Coupling of sources outside the fiber core by evanescent field excitation has been predicted by a wave model of the light interaction. Fluorescence is efficiently excited or collected only from species that are in close proximity to the sample.
If a dye is immobilized at the fiber core interface, the structure serves as a sensor for the factors affecting its fluorescence parameters. When moieties with specific binding sites are immobilized on the surface as well, the unit senses a change in the excitation of the fluorophore. The overall sensitivity of the structure depends on the modes of light propagating in the fiber, the residual thickness of the cladding, and the chemical interaction between the fluorophore and the immobilized binding sites. To gain maximum access to the evanescent field and to enhance the detection sensitivity, a portion of the fiber cladding is removed.
The invention disclosed herein is directed in at least one embodiment to an all-fiber fluorescent sensor using a relatively long, adiabatically tapered, single-mode fiber probe. The fiber is mounted in a commercial micropipette puller. The fiber is heated before and during the drawing. A carbon dioxide (CO2) laser is used as the heat source. By focusing the CO2 laser beam on the fiber and controlling the pulling force and velocity with the micropipette puller, a highly reproducible, adiabatically tapered fiber can be drawn. The tapering angle can be adjusted by changing the pulling rate, pulling force, and laser beam spot size.
Adiabatic tapering provides efficient channeling of radiation into a fiber tip region where high amplitude evanescent field will be available for excitation of fluorophores.